ABSTRACT The p53 tumor suppressor serves as one of the major cellular barriers against cancer development. p53 is controlled by its negative regulator MDM2, an E3 ubiquitin ligase. Amplification of mdm2 has been observed in many human cancers and is sufficient to induce tumorigenesis. Although only a limited number of cancer- associated mdm2 mutations have been reported, their functional studies have provided valuable information regarding the oncogenic functions of MDM2. Our previous studies have identified several novel MDM2- interacting proteins and revealed the mechanistic basis of their roles in the regulation of MDM2 function. In this proposal, we will focus on several mutant forms of MDM2 identified in tumor samples that contain a wild type p53 gene. These mutants carry MDM2 mutations within MDM2 domains previously shown to be critical for MDM2 functions. Our preliminary results indicate that these MDM2 variants have distinct functions and vary in their ability to degrade p53. In vitro transformation assays have further revealed that some MDM2 mutants have higher transformation potential than others. Moreover, we have established p53 wild-type cell lines with mutations at the endogenous mdm2 locus using TALEN-based genome editing method. Our central hypothesis is that tumor- derived MDM2 mutations deregulate the p53 function by altering p53 transcriptomes important for tumor suppression, and/or modifying MDM2 binding to its interacting proteins, which are important for the regulation of MDM2 function. This hypothesis will be tested in two independent aims using our established p53 wild-type cell lines with mutations at the endogenous mdm2 locus. In Aim 1, we will test the hypothesis that the tumor-derived MDM2 mutants alter p53 transcriptomes that are important for tumor suppression. In Aim 2, we will test the hypothesis that the MDM2 mutations modify MDM2 binding to its interacting proteins, resulting in deregulation of MDM2 function. These studies will provide a better understanding of the oncogenic activities of MDM2 and a mechanistic basis for development of novel anti-cancer strategies. In addition, this project will enhance the research environment at St. John?s University by providing undergraduate and graduate students with numerous opportunities to learn the fundamentals of biomedical research.